HPLC-TQ-MS/MS测定加味败藤浸膏中6种有效成分的血药浓度及其药动学研究

Simultaneous Determination of Six Bioactive Components of Jiawei Baiteng Extracts in Plasma by HPLC-TQ-MS/MS and Their Pharmacokinetics in Rats

目的建立HPLC-TQ-MS/MS分析方法同时测定大鼠血浆中的小檗碱、大车前苷、柴胡皂苷a、延胡索乙素、芍药苷和苦杏仁苷,并用于研究大鼠口服加味败藤浸膏后的药动学。方法色谱柱为Agilent Poroshell 120EC-C18柱(3.0 mm×100 mm,2.7μm),流动相为0.05%甲酸-乙腈(含0.05%甲酸),按程序梯度洗脱。质谱检测方式为多反应离子监测(MRM),采用正、负离子模式切换及时间分段扫描方法。用于定量分析的离子反应分别为m/z 335.9→m/z 319.9(小檗碱,ESI+),m/z 639.1→m/z 160.9(大车前苷,ESI-),m/z 779.3→m/z 617.4(柴胡皂苷a,ESI-),m/z 356.0→m/z 192.0(延胡索乙素,ESI+),m/z 449.1→m/z 121.1(芍药苷,ESI-),m/z 456.1→m/z 323.1(苦杏仁苷,ESI-),m/z 323.9→m/z 126.9(格列齐特,内标,ESI+)和m/z 321.9→m/z 170.1(格列齐特,内标,ESI-)。大鼠单剂量口服加味败藤浸膏(3.1 g·kg-1)后眼底静脉丛取血,血浆经甲醇预处理后进入HPLC-TQ-MS/MS分析。用DAS统计软件计算加味败藤浸膏中活性成分的药动学参数。结果经方法学考证,血浆中小檗碱、大车前苷、柴胡皂苷a、延胡索乙素、芍药苷和苦杏仁苷的线性范围分别为0.59~292.50,0.68~168.75,6.05~1 512.50,0.68~337.50,6.70~1 675.00和5.60~1 400.00 ng·mL^(-1),定量限分别为0.59,0.68,6.05,0.68,6.70和5.60 ng·mL^(-1),精密度、回收率及稳定性均良好,符合生物样本的分析要求。结论本实验所建HPLC-TQ-MS/MS分析方法灵敏、简便、可靠,适用于加味败藤浸膏的药动学研究。

OBJECTIVE To establish an HPLC-TQ-MS/MS assay for simultaneous determination of berberine, plantamajoside, saikosaponin a, tetrahydropalmatine, paeoniflorin and amygdalin in rat plasma and investigate the pharmacokinetics of Jiawei Baiteng extracts in rats. METHODS The separation was achieved on an Agilent Poroshell 120 EC-CI8 column（3.0 mm×100 mm,2.7μm） at 35 22. The mobile phase was consisted of 0.05% formic acid aqueous solution and acetonitrile containing 0. 05% formic acid, elu- ting with a gradient procedure. Mass spectrometry was performed in the multiple reaction monitoring （MRM） mode, with programmed ionization modes witching and time segment scanning. The ion reactions for quantification were as follows： m/z 335.9→m/z 319.9 （berberine, ESI＋ ） , m/z 639.1→m/z 160.9 （ plantamajoside, ESI- ） , m/z 779.3→m/z 617.4 （ saikosaponin a, ESI- ） , m/z 356. 0-→m/z 192.0 （ tetrahydropalmatine, ESI ＋ ）, m/z 449. 1→m/z 121.1 （ paeoniflorin, ESI - ）, m/z 456. 1→m/z 323.1 （ amygdalin, ESI- ） ,m/z 323.9→m/z 126. 9（ gliclazide, internal standard, ESI ＋ ） and m/z 321.9→m/z 170. 1 （gliclazide, internal standard, ESI- ）. Blood samples were collected in heparinized tubes via the retinal venous plexus from each rat after a single oral dose of Jiawei Baiteng extracts（3.1 g·kg^-1）. The plasma samples were pretreated by methanol precipitation to remove protein components, and then analyzed by HPLC-TQ-MS/MS. The pharmacokinetic parameters of the bioactive components of Jiawei Baitengex tracts in rats were calculated by DAS （Drug and Statistics for Windows） software（Version 2. 0）. RESULTS The methodological test showed that the linear concentration ranges of berberine, plantamajoside, saikosaponin a, tetrahydropalmatine, paeoniflorin and amygdalin were 0. 59 -292. 50, 0. 68 - 168.75, 6. 05 - 1 512. 50, 0. 68 -337. 50, 6. 70 - 1 675.00 and 5. 60 - 1 400. 00 ng·mL^-1, respectively. The limits of quantification （ LOQs ） of the six analytes were 0. 59,0. 68,6. 05,0. 68,6. 70 and 5. 60 ng·mL^- 1, respectively. The HPLC-TQ-MS/MS method was also validated with good precision, recovery and stability, which conformed to the analytical standards of biological samples. CONCLUSION The established method is proved to be sensitive, simple and reliable, which is suitable for the pharmacokinetic study of Jiawei Baiteng extracts.